The objective of this proposal is to study protein folding using single-molecule fluorescence methodology. Understanding protein folding is fundamental to deciphering how the genetic code is translated into functional protein units. Improper protein folding is related to complex aggregation phenomena that cause diseases such to the prion family of diseases and Alzheimer's disease. We will develop single-molecule fluorescence methodology to follow conformational dynamics of individual fluorescently-labeled proteins and other biopolymers. In contrast to ensemble methods, single-molecule methods provide information on fluctuations, distributions and time-trajectories of observables that are obscured in ensemble measurements. In particular, single-molecule methods are free from synchronization requirements that are impossible to achieve with ensemble methods. We will use single-pair fluorescence resonance energy transfer (spFRET) as reporter of the distance between two amino acid residues in the polypeptide chain, or the site-to-site distance of a fluctuating biopolymer. The recovered distance information will be used as a reaction coordinate of protein folding, and as a reporter of conformational dynamics. We will emphasize early events in the folding pathway, especially focusing on the "fast-collapse" of the polypeptide chain when exposed to specific solvent conditions. We will address both equilibrium and non-equilibrium reaction conditions, and study molecules that are (i) diffusing in solution, or (ii) immobilized on surfaces and in gels. We will develop single-molecule, continuous flow, fast-mixing methods (for diffusing molecules) and rapid liquid-exchange methods (for immobilized molecules). Our measurements will combine fluorescence-intensity ratiometric methods with fluorescence-lifetime methods to provide unprecedented sensitivity and temporal resolution that cover many orders of magnitude. Advanced data acquisition, data analysis and signal processing algorithms will be developed for a variety of samples, experimental formats and reaction conditions. A theoretical framework will be constructed to accurately describe the results of our experiments. We will: (i) develop single-molecule methods with spatial and temporal resolution relevant to protein folding; and (ii) use homopolymeric single-stranded DNA (ssDNA) as a model system for method development. We will combine the proposed methodology with existing single-molecule fluorescence methodology to study the folding of protein chymotrypsin inhibitor 2 (CI2). In particular, we will address: (1) early events of the folding pathway, such as fast collapse; (2) late events of the folding pathway, such as formation of native protein contacts; and (3) effects of amino acid substitutions on early and late events of the folding pathway.